The long-term objectives of this application are 1) to analyze the molecular structure and mechanism of action of the human mitochondrial transcription termination factor (mTERF), its regulation and its role in human diseases; 2) to use the cellular models of mtDNA-linked diseases developed in the laboratory, utilizing human mitochondrial DNA-less cells as recipients for mitochondrial transformation, to answer basic questions concerning the biochemical mechanism of disease-causing mutations, the basis for the tissue specificity of the mutation-associated phenotype and the dynamics of complementation and segregation of the mutations; and 3) to elucidate the physiological role of the mitochondrial DNA-encoded subunits of the respiratory NADH dehydrogenase. Specifically, the present proposal aims at identifying the components required for making mTERF competent for transcription termination, at reconstructing in vitro the termination event with purified components, and at determining by x-ray crystallography and circular dichroism and NMR measurements the three-dimensional structure of mTERF alone and associated with DNA. Furthermore, it is planned to clone the nuclear gene of the human mTERF, to study the regulation of its expression, and to knock out the mTERF homologous gene in the mouse and to investigate the developmental and physiological effects of this gene disruption. It is also planned to investigate in human cells in culture the factors that control the complementation, selection and segregation of mutant mtDNA and wild-type mtDNA coexisting in the same cell including the role of the nuclear background. Finally, it is planned to analyze the role of nucleotide modifications of tRNAs and of changes in the flux control of respiration in the pathogenic mechanisms of these mutations. The achievement of the above aims may have significant implications for understanding the regulation of mitochondrial gene expression, for elucidating the role of the mitochondrial genome in controlling the function of a key enzyme in the cell energetic metabolism, as well as for understanding some fundamental features of mammalian mitochondrial genetics. In another context, these studies are expected to provide insights into the pathogenic mechanisms of mtDNA mutations causing disease in man, into the establishment and transmission of these mutations and in general, into the inheritance and evolution of mtDNA.